PROJECT SUMMARY/ABSTRACT Single-cell analysis of cell states carries tremendous potential for biological inquiry and biomedical applications. For example, a cell's functional state may explain heterogeneous cell responses to environmental ligands or to drugs, while an accurate catalog of the heterogeneous cell states in a tumor may ultimately prevent incomplete drug response and drug resistance development. Although current sequencing methods (i.e. scRNA-seq) offer transcriptional signatures of single cells, a cell's functional state is more accurately described by its proteomic signature, for which the transcriptome is only weakly predictive. However, there are no methods to measure single cell proteomes with comparable sensitivity and throughput to scRNAseq because proteins cannot be amplified and sequenced like nucleic acids. Our long-term goal is to address this gap through developing scProteome-seq, a technology to quantify 102-103 proteins and post-translational modifications (PTMs) from ~104-105 individual cells simultaneously. scProteome-seq achieves specificity in protein detection because it first uses single-cell western blots (scWesterns) to separate a cell's protein content by size in a sieving gel prior to antibody probing. Sensitivity and high-throughput analysis are achieved via separate DNA barcodes encoding a protein's location (encoding protein size and cell ID) and identity (encoding antibody identity). The two barcodes are linked, extracted, and subsequently quantified by PCR amplification and sequencing. In this proposal, we address the feasibility of key technological challenges to this approach in two aims. In Aim 1, we engineer methods to spatially barcode scWestern separations gels with DNA. First, strategies for printing and covalently attaching DNA onto gels will be developed and analyzed for efficiency and spatial resolution. Second, DNA-modified scWestern gels will be validated for separations performance compared to unmodified gels. In Aim 2, we develop the barcoding strategy for scProteome-seq. Barcode sequences will be designed to enable physical coupling, gel-extraction, and amplification. Specificity and efficiency of coupling will be optimized, and protocols for barcoding, coupling, extraction, and analysis will be developed. Successful completion of these aims will form the foundation for full-scale development, quantitative characterization, and ultimate deployment of scProteome-seq. We anticipate that proteomic fingerprinting of cell states will provide significant technical advances applicable across a broad range of fundamental and translational biomedical disciplines.